Sensitive methods exist to detect target molecules such as particular nucleic acids, proteins or more simple molecules. The presence of such molecules may be used to indicate an on-going infection or environmental contamination, for example. In prion diseases it would be useful to be able to detect the prion protein where no nucleic acid is present. Also, at certain stages of a viral infection there will be virus antigen present but little viral nucleic acid present. Here it will be useful to be able to detect the viral antigen directly. In order for these methods to be very sensitive and to detect as little as a single molecule the methods must also have high specificity. This high specificity is often achieved by binding two reporters to the target molecule that is to be detected.
In the case of the highly sensitive polymerase chain reaction (PCR), for example, two short nucleic acid probes or primers recognise the target nucleic acid. The detection of the target nucleic acid is thus only achieved when both primers are bound to, and linked through, the same target molecule. Non-specific interactions of the primers with other molecules are not detected unless both primers bind to and are linked by this non-specific interaction. The conditions of the reaction are such that the latter is highly unlikely. The PCR method and other molecular amplification methods, well known in the art, such as Nucleic acid sequence-based amplification (NASBA; Compton, 1991)(1), Transcription Mediated Amplification (TMA; Gen-probe, Inc.) and Self-sustained sequence replication (3SR; Fahy et al., 1991)(2) can be used to detect target nucleic acids.
Immunoassays are often employed in order to detect specific analytes/antigens of interest. Here an antibody, usually a monoclonal antibody, is used in order to allow specific detection of the analyte/antigen. Immuno detection methods can be broadly split into two main categories; solution-based techniques such as enzyme-linked immunosorbent assays (ELISA), immunoprecipitation and immunodiffusion, and procedures such as Western blotting and dot blotting where the samples have been immobilized on a solid support.
Western blot analysis relies on a primary antibody directed against the antigen/analyte, which is added to a membrane containing immobilized antigen/analyte to allow binding to potential antigenic sites. Next, a secondary antibody-enzyme conjugate which recognizes the primary antibody is added in order to find locations where the primary antibody bound. The enzyme, commonly alkaline phosphatase or horseradish peroxidase, conjugated to the secondary antibody can catalyze a reaction with a chemiluminescent substrate in the third step leading to emission of light from the membrane at the reaction site. An x-ray film exposed to the signal provides a visual indication of potential primary antibody recognition. The action of horseradish peroxidase or alkaline phosphatase on a chemiluminescent substrate can give sensitivity down to the picomolar range. Antigens/analytes can be immobilized on nitrocellulose or polyvinylidene fluoride (PVDF) membranes by numerous methods. The ability to detect a given antigen/analyte depends upon the amount of antigen per unit area of the membrane and on the characteristics of the primary antibody.
ELISAs provide sensitive and quantitative detection of specific antigens/analytes. The most common ELISAs are based on an antibody-sandwich format. A sandwich ELISA generally requires two antibodies that are directed against a particular antigen. One antibody is coated onto the wells of the ELISA plate. The wells are then “blocked” using a non specific protein solution (such as milk protein solution) to keep background levels down to a minimum. Samples containing the antigen in solution are then added to the wells and incubated for a sufficient amount of time to allow antigen binding to the immobilized antibody. The second antibody can then bind to the antigen to complete the “sandwich”. The second antibody is detected with an enzyme conjugate specific for the second antibody. As an alternative, the second antibody can be labeled itself to allow subsequent detection. When the enzyme substrate is added to the wells in the final step, the conjugated enzyme, which is linked to the antigen, is detected by observing a reaction product which may be calorimetric, fluorescent or chemiluminescent depending on the enzyme and substrate used, using an ELISA plate reader.
The most commonly employed enzymes in immunoassays are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Such enzymes can react with a substrate chromogen to give a coloured product in the presence of an antigen. For example, a substrate chromogen commonly used in conjunction with alkaline phosphatase is 5-bromo, 4-chloro, 3-indolylphosphate (BCIP). An additive such as iodoblue tetrazolium (INT) may also be used to enhance the final colour of the precipitate at the reaction sites, that is where the primary and secondary antibodies have bound to the antigen (which would be a yellow-brown colour for BCIP with INT).
Alkaline phosphatase also has the ability to remove 5′ phosphate groups from DNA and RNA. It can also remove phosphates from nucleotides and proteins. These enzymes are most active at alkaline pH. Three major types are commonly employed in immunoassays. Bacterial alkaline phosphatase (BAP) is a highly active enzyme. Calf intestinal alkaline phosphatase (CIP) is purified from bovine intestine, and can be inactivated using protease digestion or heat, for example. Shrimp alkaline phosphatase is derived from a cold-water shrimp and can be inactivated using heat treatment fairly readily.
HRP can be used in a number of bioassays. Peroxidase activity is also present in many cells. Many fluorogenic substrates for HRP are well known in the art and are commercially available. One example is Amplex Red Reagent (Molecular Probes), 10-acetyl-3,7-dihydroxyphenoxazine, which can react with H2O2 in a 1:1 stochiometry in the presence of HRP to produce highly fluorescent resorufin. An alternative substrate is scopoletin, where HRP catalyzes conversion of the fluorescent scopoletin to a nonfluorescent product. Such substrates are commonly included in ELISA kits to allow detection of sites where an antigen/analyte is present.
Numerous attempts have been made to combine the advantages of immunoassays and nucleic acid amplification techniques. Indirect conjugation methods may be used to link a protein to a nucleic acid molecule. For example, an enzyme such as alkaline phosphatase may be covalently bound to a molecule such as biotin and digoxigenin. This conjugate in turn can then be non-covalently attached to a biotinylated nucleic acid probe via a streptavidin bridge, to be used, for example in Southern and Northern blotting techniques. Such methods can produce consistent results, however the protocols can take much longer than those of direct conjugation methods. Usually several incubation and washing steps are required to bind additional bridging molecules such as streptavidin or an antibody to the labeled probe before the enzyme and substrate can be introduced. Furthermore, with each additional step there is an increased chance of adding background to the signal.
Thus, direct conjugation of an enzyme to a probe is a preferable option, to increase speed and maximise sensitivity. Alkaline phosphatase-conjugated oligonucleotides (Sigma-Genosys) can be used for routine screening applications such as Southern (DNA) and Northern (RNA) blotting, gene mapping and restriction fragment length polymorphism (RFLP) analysis. They can also be used for in situ hybridizations.
Enzyme immunoassays have been established as the most ubiquitous methods for detection of antigen. They are simple, robust and easy to perform. In those cases where extra sensitivity is required more complex and expensive nucleic acid amplification tests such as the Polymerase Chain Reaction (PCR) can be performed.
Numerous attempts have been made to combine the advantages of both approaches. For example, there is use for a sensitive nucleic acid test that can detect antigen. This would be useful in prion detection where there is no associated nucleic acid or in blood bank screening where, at certain times post-infection, there can be virus antigen but little viral nucleic acid.
Previous attempts to combine the immuno and nucleic acid approach by using antibodies labeled with nucleic acids (so-called immuno PCR) have had problems. Linking DNA to antibodies is problematical and the linked DNA is ‘sticky’ and any unbound DNA is not easily washed from the system prior to detection which can lead to non-specific binding and a high background in the assay.
WO 2005/012567 relates to methods for detecting an enzyme in a sample which is capable of modifying a nucleic acid molecule by detecting the change in the nucleic acid molecule caused by the enzyme. The specific methods disclosed all relate to protection of the nucleic acid molecule from digestion and thus do not detect formation of a new nucleic acid molecule. Thus, the methods, whilst being highly sensitive and efficient methods for detecting a phosphatase enzyme, have as a disadvantage the fact that false positive results may be obtained where the digestion is not 100% efficient. Additionally, if phosphate labelling of the nucleic acid molecule is likewise not 100% efficient this can similarly lead to false positive results.
The present invention overcomes the problems associated with prior art methods as described below.